人羊水来源干细胞以类胚体样结构形式向心肌样细胞分化的可行性  

Cardio-myogenic differentiation of human amniotic fluid colony derived stem cells in the form of embryonic body-like structure

作　　者：马晓荣[1] 谢华[1] 张胜利[1] 高同斌[1] 王雪[1] 尚亚峰[1] 周君梅[2] 陈方[1] 

机构地区：[1]上海交通大学附属上海市儿童医院小儿泌尿外科,200040 [2]上海交通大学附属上海市儿童医院中心实验室,200040

出　　处：《中华医学杂志》2011年第46期3293-3297,共5页National Medical Journal of China

基　　金：基金项目：上海市科学技术委员会科研基金（08JC1416000）;上海市教育委员会科研创新项目（09YZ90）

摘　　要：目的 探讨人羊水来源干细在体外以类胚体形式向心肌样细胞分化的可行性.方法 在B型超声引导下穿刺抽得孕中期羊水,体外分离、培养得到羊水来源干细胞.取第四代细胞消化计数,悬滴法培养5d后,采用逆转录-聚合酶链式反应（RT-PCR）检测干细胞相关mRNA表达情况；更换诱导培养液以贴壁培养方法诱导类胚体细胞向心肌样细胞分化2周.RT-PCR、免疫荧光染色方法鉴定心肌细胞特异mRNA及蛋白的表达.结果人羊水来源干细胞体外悬滴培养后24h即可形成类胚体样结构；类胚体样结构直径随培养时间逐渐增大,第5天时,类胚体样结构平均直径为（237.3±26.7）μm,形成率为93.5％；RT-PCR检测到干细胞相关mRNA表达；类胚体形式下,经诱导后,免疫荧光染色检测类胚体中羊水来源干细胞表达心肌细胞部分表型：肌结蛋白（Desmin）、肌钙蛋白Ⅰ（TnⅠ）和横纹肌辅肌动蛋白（α-Actinin）；RT-PCR检测类胚体中羊水来源干细胞表达心肌细胞相关mRNA：肌结蛋白（Desmin）、肌动蛋白（α-Actin）、肌钙蛋白I（TnⅠ）、肌钙蛋白T（Tn T）、T-box、NK2.5.而对照组呈阴性.结论人羊水来源干细胞体外悬滴培养后可形成形态均一的类胚体样结构.5-氮杂脱氧胞苷（5-Aza）能诱导类胚体样结构中的羊水来源干细胞向心肌样细胞分化.Objective To investigate cardio-myogenic differentiation potential of human amniotic fluid colony derived stem ceils（HAFCDSC）in the form of embryonic body（EB）-like structure in vitro.Methods HAFCDSC were isolated from second trimester amniotic fluid which was backup of amniocentesis specimens.The forth passage of HAFCDSC were cultured by hanging-drop preparation in complete medium for 5 days to form EB-like structures followed by inducing medium in regular tissue culture dishes for 2 weeks（experiment group）.The EB-like structures cultured in complete medium were served as conrol group.Cardiomyocyte phenotypes were detected by RT-PCR and immunofluorescence staining.Results HAFCDSC could form EB-like structures 24 h after hanging drop culture,and the diameter of EB-like structures gradually increased with culture duration and the mean diameter of EB-like structures reached 237.3-26.7 μn on 5th day.The formation rate of EB-like structures was 93.5%.RT-PCR analysis showed EB-like structures expressed stem cell related mRNA.Immunofluorescence staining demonstrated that some cardiomyocyte phenotypes such as Desmin,t-Actinin and Tn I were expressed by HAFCDSCs in EB-like structures after cardio-myogenic induction.Cardiomyocytes specific mRNA includingDesmin,t-Actin,Tn Ⅰ,Tn T,T-box and NK2.5 were also transcripted as detectd by RT-PCR.No positive results was found in control group.Conclusion HAFCDSC can form uniform EB-like structures after hanging drop culture for 5 days.HAFCDSC differentiate into cardiomyocyte-like cells through the form of EB-like structures after induction by 5-Aza.

关 键 词：干细胞 类胚体 心肌细胞 分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]